The purpose of this project is to investigate the structure and function of membrane-associated proteases. Both the cytoplasmic and outer membranes of E. coli contain proteases. The outer membrane protease can be measured in vitro by solubilization of nitrate reductase from membrane vesicles and the protease in the cytoplasmic membrane by hydrolysis of the artificial substrates BAPN and hide powder. All of these proteases are active in the presence of detergents and will be purified after detergent solubilization. They will be characterized in reference to enzymatic activity, in vivo function, structure, location within the membrane and substrate interactions. The responses of these proteases to a group of protease inhibitors will be tested. The specificity of these protease inhibitors will be correlated with in vivo actions of these inhibitors. To do this, cells will be grown in varying amounts of each inhibitor and examined for differences in proteins in the cytoplasm, cytoplasmic membrane and outer membrane. In vivo studies on the turnover of nitrate reductase will also be carried out. The biosynthesis and turnover of normal and abnormal nitrate reductase will be followed under various physiological conditions.